Conventional methods of detection and isolation of proteins and other molecules have employed antibodies and the like which specifically bind such substances. Recently, however, the de novo design of specifically binding oligonucleotides for non-oligonucleotide targets that generally bind nucleic acids has been described. See, e.g., Blackwell, T. K., et al., Science (1990) 250:1104-1110; Blackwell, T. K., et al., Science (1990) 250:1149-1152; Tuerk, C., and Gold, L., Science (1990) 249:505-510; Joyce, G. F., Gene (1989) 82:83-87. Such oligonucleotides have been termed "aptamers" herein. Tuerk and Gold describe the use of a procedure termed "systematic evolution of ligands by exponential enrichment." In this method, a pool of RNAs that are completely randomized at specific positions is subjected to selection for binding by a desired nucleic acid-binding protein which has been fixed on a nitrocellulose filter. The bound RNAs then are recovered and amplified as double-stranded DNA that is competent for subsequent in vitro transcription. The newly transcribed RNA then is recycled through this procedure to enrich for oligonucleotides that have consensus sequences for binding by the cognate protein. The oligonucleotides so obtained then may be sequenced for further study. Tuerk and Gold applied this procedure to identify RNA oligonucleotides which are bound by the RNA binding region of T4 DNA polymerase.
Kinzler, K. W., et al., Nucleic Acids Res. (1989) 17:3645-3653, applied this technique to identify double-stranded DNA sequences that were bound by proteins that bind to DNA and regulate gene expression. In the reported work, total genomic DNA is first converted to a form that is suitable for amplification by PCR by ligation of linker sequences to the genomic DNA fragments and the DNA sequences of interest are selected by binding mediated by the target regulatory protein. The recovered bound sequences are then amplified by PCR. The process of binding by protein and amplification are repeated as needed. The selection and amplification process are repeated as needed. The process as described was applied to identify DNA sequences which bind to the Xenopus laevis transcription factor 3A. The same authors (Kinzler et al.) in a later paper, Mol. Cell Biol. (1990) 10:634-642, applied this same technique to identify the portion of the human genome which is bound by the GLI gene product produced as a recombinant fusion protein. The GLI gene is amplified in a subset of human tumors.
Ellington, A. D., et al., Nature (1990) 346: 818-822, describe the production of a large number of random sequence RNA molecules and identification of those which bind specifically to immobilized target molecules, in the case of this paper, to specific dyes such as Cibacron blue. Randomly synthesized DNA yielding approximately 10.sup.15 individual sequences was amplified by PCR and transcribed into RNA. It was thought that the complexity of the pool was reduced in the amplification/transcription steps to approximately 10.sup.13 different sequences. The pool was then applied to an affinity column containing the dye and the bound sequences subsequently eluted, treated with reverse transcriptase and amplified by PCR. The results showed that about one in 10.sup.10 random sequence RNA molecules folds in such a way as to bind specifically to the ligand.
Thiesen, H.-J., and Bach, C., Nucleic Acids Res. (1990) 18:3203-3208, describe what they call a target detection assay (TDA) to determine double-stranded DNA binding sites for putative DNA binding proteins. In their approach, a purified functionally active DNA binding protein and a pool of random double-stranded oligonucleotides which contain PCR primer sites at each end were incubated with the protein. The resulting DNA complexes with the protein (in their case, the SP-1 regulatory protein) were separated from the unbound oligomers in the random mixture by band-shift electrophoresis and the SP-1 bound oligonucleotides were rescued by PCR and cloned, and then sequenced.
Copending U.S. application Ser. No. 07/586,769, filed Sep. 21, 1990, the entire disclosure of which is hereby incorporated by reference, describes a method utilizing a binding site selection technique which depends on the availability of PCR (polymerase chain reaction). In this approach, selected and amplified oligonucleotides were used to identify consensus DNA recognition sequences used by DNA binding proteins under study.
None of the above references, however, describes a method in which the aptamer DNA-target complexes are isolated by coupling the target to a column support, and then purified by uncoupling the target-aptamer complex.
The invention herein provides such an approach and utilizes a binding site selection method combined with PCR to develop aptamers with selected and amplified binding sites. In a preferred embodiment this process is aided by consensus sequences.